This invention relates to processes for carrying out specific binding assays, e.g. immunoassays, and to apparatus, e.g. test kits, for carrying out these processes.
In particular embodiments the invention is applicable to enzyme-linked and fluorescent-marker-linked specific binding assays, including immunoassays.
A wide variety of immunoassays and other specific binding assays is already known.
Examples of such assays and the materials used for them are given in S. Spector, Ann.Rev.Pharm. (1973) 13, 359-70 (radioimmunoassays), L. E. M. Miles and C. N. Hales, Nature (1968) 219, 186-189 (assays using radioactive, enzyme and other markers), E. Habermann, z.klin.Chem.u.klin.Biochem. (1970) 8, 51-55 (radioactively-labelled and enzyme-labelled assay procedures), and in G.B. patent specification No. 1,363,565 (enzyme-labelled immunoassays) and U.S. Pat. Nos. 4,150,949 and 4,160,818 (fluorescence-labelled immunoassay).
An important group of such assays comprises those in which a ternary complex is formed between a specific adsorbent, the material under assay, and a marker-conjugated material with specific binding capacity for the material under assay (xe2x80x9cconjugatexe2x80x9d). Certain assays of this kind have been called xe2x80x9csandwichxe2x80x9d or xe2x80x9cantiglobulinxe2x80x9d assays. They have the property that the quantity of marker becoming fixed to the specific adsorbent is directly rather than inversely related to the quantity of the material under assay that participates in the ternary complex, and this can be simply measured after separation of the immobilised material from the remaining free marked conjugate, in whatever manner is appropriate to the marker in use.
However, as appears for example from the above-mentioned Habermann (1970) publication and G.B. specification No. 1,363,565, the performance of these assays is not without difficulty: they demand either a number of successive manipulation steps to carry out the assay reactions, or else suffer from low sensitivity, which careful choice of reagents has not so far been able to overcome.
Commercial test kits are available comprising essentially:
(a) a plate consisting of an array of tubes or pre-formed wells which are coated with an antibody or antigen as the case may be;
(b) an enzyme linked to an appropriate antibody (a so-called conjugate) against a substance to be detected if present in a test sample and
(c) a substrate for the determination of the activity of the enzyme.
One standard procedure for conducting an assay for antigen or antibody involves:
(1) determining the working dilution for the test sample;
(2) removing any excess of antibody or antigen used to sensitise the wells;
(3) washing the wells;
(4) introducing a proportion of the suitably diluted test sample;
(5) incubating for about two hours to allow the substance to be detected in the test sample to bind to the sensitising substance;
(6) washing the wells to remove unreacted material;
(7) introducing the suitably diluted conjugate (incubate for about 2 hours);
(8) washing the wells to remove unreacted material;
(9) adding a solution of the substrate;
(10) incubating until a suitable intensity of colour develops as a result of the reaction of the substrate with the enzyme;
(11) stopping the reaction, e.g. with a strong alkali and
(12) measuring the optical density of the reacted substrate solution.
This procedure is also time-consuming since each of the several antibody-antigen reactions requires several hours to reach equilibrium. In practice, shorter incubation times are used but only at the expense of sensitivity and/or economy.
According to the results of the present work, it is believed that an obstacle to the use of fewer assay steps is an unwanted interference with the formation of the desired immobilised complex which can originate in interfering reactions between two of the components. By using a specific binding agent of selected narrow specificity, or in slow-release form, such interference can be avoided, and high-sensitivity assays carried out using fewer manipulation steps.
According to this invention there is provided a process for carrying out a specific binding assay (for example an immunoassay) in which (a) a sample under assay, possibly containing a substance being tested for, is reacted with (b) a specific binding partner for the substance being tested for, immobilised on a solid support, and (c) a specific binding partner for the substance being tested for which is conjugated to a detectable marker, thereby to form a complex by reaction between whatever quantities are present of the substance being tested for with reagents (b) and (c), in which the marker is immobilised to the support via the substance being tested for, and is detected or assayed as an index of the quantity present in the sample (a) of any of the substance being tested for; characterised in that reaction ingredients (a), (b) and (c) are all mixed in a single step for reaction in a single reaction liquid, and competitive interference between the binding reactions of the substance being tested for and reagents (b) and (c) is avoided either by use of an antibody of narrow specificity, such as a monoclonal antibody, to avoid the interference, or by use of a slow-release form of reagent (c).
The narrow specificity required of the antibody is a capacity to bind specifically with the substance under test but without preventing the binding reaction between the substance under test and its other specific binding partner. Such an antibody can be selected out of a number of antibodies with an affinity for the substance under test, by using normal methods to verify the progress of a binding reaction between the other specific binding partner and a complex previously formed between the substance to be tested in the assay and the narrow-specificity antibody to be selected.
According to a preferred embodiment of the present invention, the conjugate between antibody and the enzyme or other marker, and/or the antibody (if any) which is coupled to the solid surface, comprises a monoclonal antibody or other antibody of sufficiently narrow specificity to ensure that the desired assay reaction or reactions are not impeded by competition between the conjugate and the immunosorbent in their reactions with whatever quantities are present of the substance being tested for in the sample under assay. Monoclonal antibody of sufficiently narrow specificity can, for example, be produced as antibody derived from a line of antibody-producing cells, derived from a single antibody-producing progenitor cell or cells. Such a line can, for example, be produced by known cell fusion, culture and isolation techniques using very pure antigens as comparative material.
Alternatively, in many cases antibody of sufficiently narrow specificity can be obtained in the (polyclonal) immunoglobulins of antisera raised against discrete chemical or physical molecular fragments of the material under test, for example, antibody against Fc fragments (or against smaller peptide fragments) of immunoglobulins to be tested for, or against sub-units or peptides of protein antigens to be tested for. The object in each case is to ensure substantial freedom from interference which can arise particularly, for example, in carrying out immunoassays of the xe2x80x9csandwichxe2x80x9d or xe2x80x9cantiglobulinxe2x80x9d test configurations.
In a xe2x80x9csandwichxe2x80x9d test configuration, antigen under test can be specifically adsorbed to a first antibody bound to a solid surface, and a second antibody carrying an enzymic or other (e.g. fluorescent or radioactive) marker is specifically bound to the adsorbed antigen under test. Marker specifically so bound is used for measurement and determination of the antigen under test, e.g. by direct measurement, such as radiometry or fluorimetry, or exposure of enzyme marker to substrate followed by product measurement. Thus, in preferred sandwich tests, the two antibodies used can have different, non-interfering specificity with respect to the same antigen under test.
In an xe2x80x9cantiglobulinxe2x80x9d test configuration, sometimes also referred to as a xe2x80x9csandwichxe2x80x9d test configuration, the position is analogous: the material under test is itself an immunoglobulin; the material bound to a solid surface is its corresponding antigen or hapten; and the material carrying the marker is an antiglobulin corresponding to the species and immunoglobulin type of the antibody under test. In preferred antiglobulin tests, the antiglobulin can have sufficiently narrow specificity as not to interfere with the subsequent adsorption of its corresponding globulin to the insolubilised antigen.
If antibodies from ordinary antisera raised against unmodified antigen (polyclonal antibodies) are used in sandwich or antiglobulin tests, there is a very likely risk that if all ingredients are mixed in a single step there will be interference between the two specific adsorption reactions. When such tests are carried out according to the present invention, using apparatus as described herein, such interference can be avoided either by using antibodies of narrow specificity as described, or else by ensuring that the binding of test material to the solid surface takes place before exposure of test material to the other (marker-conjugated) binding agent if there is a risk that binding by that other agent would prevent subsequent adsorption to the solid surface. Such a sequence can be ensured by arranging for slow release of the other (marker-conjugated) binding agent.
Particular instances of suitable assay specificities, antibody specificities, and slow-release forms of conjugated reagent (c) are described for example below.
It has also been found that in carrying out such specific binding assays, a worthwhile improvement in reaction kinetics can be obtained if the reaction liquid containing ingredients (a), (b) and (c) is contained in a well or cup of which the majority of the volume is occupied by a displacer body. (The use of inserts of various rod or ball shaped forms is known in connection with other kinds of immunoassay, as described in G.B. Specification Nos. 1,414,479 and 1,485,729.)
The displacer body can, for example, be of a shape substantially complementary to and slightly smaller than that of the cup or well, so that the liquid phase containing one of the specific binding reagents is approximately in the form of a shell occupying the space between the displacer and the cup or well. The displacer can be loose-fitting and not fixedly mounted, i.e. movable relatively to the cup or well, so that by relative motion between displacer and well the liquid between them can be given a stirring or agitation motion.
For example, a round well can have a round displacer therein with an external diameter slightly smaller than the diameter of the well. The presence of the displacer can reduce the space available for liquid in the well by a factor of for example 2-10, e.g. 3-8, comparing volumes based on similar liquid levels in the well, e.g. when filled to its normal operating level, or its maximum capacity. For example, a microtitre well designed to have 300 microlitre of liquid filled into it during a normal assay, can be used with a displacer leaving 30-150 microlitre liquid space, e.g. 50-100microlitre.
The use of wells or cups together with displacers as described herein can improve the efficiency of the assay reaction steps because, in the first place, it allows more concentrated reagents to be used with no increase in the weight of reagent or decrease in the size of the microtitre wells, compared with the normal conditions encountered in microtitre wells of given size; and in the second place, it increases the sensitised surface area available to react with a given liquid reagent volume, so that comparatively faster adsorption kinetics can be achieved without having to increase specific reagent density on the sensitised surface or encountering problems of crowding.
A set of displacer bodies can be preferably present in certain embodiments of the invention, e.g. as an integral part of a lid which can be fitted onto a microtitre plate, e.g. a standard plate of 8xc3x9712 wells. The set can be large enough to fit all the wells of the plate or a sub-set thereof, e.g. a row. The dimensions of the displacers and the volume of liquid to be dispensed into the well can be chosen relative to the well in the manner described above, and preferably so that the liquid to be tested is in contact with substantially the major part and preferably the whole inner surface of the well.
The immobilised specific binding partner (reagent (b)) for the substance to be assayed can be immobilised on the wall of the well or cup in which the assay reaction takes place. Alternatively, according to a feature of the invention independently capable of providing advantage and convenience in use, a liquid displacer, for example in the form of a stick, peg or stud, for dipping into a liquid assay reagent, can have an immunosorbent surface. This allows the portion of the assay materials needing to be carried over from one reagent to the next, and the associated manipulations, to be handled more easily than when the sensitised surface is part of a hollow well. An alternative form for such a liquid displacer body is a tuft of bristles or leaves of suitable material, or equivalent body with large surface area. A further alternative form is a stud or peg with relatively hollowed-out and projecting portions of its surface, e.g. with grooves and associated ribs, e.g. annular grooves. Such an arrangement can give robustness, increased sensitised surface area, and better reactivity.
Test apparatus according to related embodiments of the invention can thus comprise a set of sensitised liquid-displacer bodies of one or more of such forms, joined to a common handling-bar, link or lid, and for use in combination with a complementary set or sets of wells containing any of the remaining materials used in the assay. The several displacer bodies of the set can have the same or different sensitisation so that one or a plurality of different assay types can be carried through simultaneously. If desired, the displacer bodies can be removably and exchangeably mounted on the handling bar, link, or lid, so that sets of desired specificity can be built up at will from a common stock for carrying out large numbers of tests according to a desired pattern.
One advantage of such arrangements is that a number of displacer bodies can be sensitised in the same body of liquid ragent, avoiding fluctuating conditions of concentration, etc., resulting from dosing aliquots into wells.
The displacer bodies can, in this embodiment, be of any material suitable for the preparation of an immunosorbent by covalent bonding or adsorption: e.g. polystyrene, nylon, or cellulose acetate. (The nature of the displacer surface does not matter provided it is inert, when the sensitisation is to be on the well surface rather than the displacer surface.) Linkage of antibodies, antigens, etc., to the displacer bodies can be carried out by linking methods known in themselves, e.g. partial acid hydrolysis of nylon surface, substitution of exposed amine surface with glutaraldehyde, and coupling of material to be bound, e.g. antibody or antigen, to immobilised aldehyde groups. Suitable methods among a wide variety are given for example by Inman and Hornby (1972) Biochem.J. 129, 255; Campbell, Hornby and Morris (1975) Biochim,Biophys.Acta 384, 307; Mattiasson and Nilsson (1977) F.E.B.S. Letters 78, 251; and G.B. patent specifications Nos. 1,470,955 and 1,485,122.
It can be seen that the invention also provides a kit of test materials for carrying out a specific protein-binding assay, comprising (i) an immobilised specific binding partner for a substance to be tested for, carried on a solid support, and (ii) a marker-conjugated specific binding partner for the substance to be tested for, which can be added to a reaction liquid contacting immobilised reagent (i) either as a slow-release form, or in any form provided that the specific binding partners in reagents (i) and (ii) include an antibody of narrow specificity so that reagents (i) and (ii) do not interfere with each other""s binding reactions with the substance to be tested. Optionally the kit can also comprise materials for later estimation of the amount of marker immobilised during the assay.
Reagent (i) can be immobilised on either a displacer body for a reaction well, or on a reaction well wall, as described above. A slow-release form of reagent (ii) can be for example a sucrose or equivalent glaze on a complementary surface of either the displacer or the well wall, also as described above. The narrow-specificity antibody can be selected for example from monoclonal antibodies in the manner already described.